Temperature is an important stimulus, and animals including nematode C. elegans have habituation mechanisms to environmental temperature. We are using temperature experience-dependent cold tolerance of C. elegans, as a model for studying temperature sensation and memory. After cultivation at 20°C, wild-type were destroyed by cold stimuli. By contrast, after cultivation at 15°C, most of animals can survive. Recently, we showed that head sensoryneuron ASJ senses temperature through trimeric G-protein signaling, and ASJ releases insulin that regulates cold habituation by expressing downstream genes. To isolate new genes involved in the cold tolerance, we are using three approaches, (1) Artificial evolution, (2) EMS-mutagenesis and (3) DNA microarray analysis.
(1) C. elegans has strong advantage for artificial evolution analysis, since life cycle is short and strains can be preserved at -80°C. C. elegans has strong advantage for artificial evolution analysis. We are maintaining C. elegans at 15 or 23 degrees for gradually accumulating spontaneous mutations, and frozen-stocks of animals were made at every generation. We so far obtained about 110 generations animals, and cold tolerance phenotypes have been gradually changed. We are planning to decode the genomes of frozen-animals by using deep DNA sequencer.
(2) We isolated several mutants defective in cold tolerance from two thousands genomes screening by EMS mutagenesis. KHR1 showed significant decrement of cold habituation. To isolate responsible genes for KHR1, we utilized single nucleotide polymorphism (SNP) in wild-type strains isolated from different areas, Bristol and Hawaii. So far, Responsible gene of cold tolerance in KHR1 strain was mapped on chromosome X -7.48 cM~-0.83 cM. We are now analyzing detailed-mapping position by using classical genetics and deep DNA sequencer.
(3) To isolate downstream molecules of insulin signaling for cold habituation, we performed DNA microarray analysis comparing between wild-type and insulin signaling mutant. We found that mutants impairing 15 genes, such as protein phosphatase (PP1), laminin, showed abnormal cold habituation. We are now analyzing responsible cells for the abnormalities of these mutants.